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Results for “protein purification”

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At least 181 records · Page 10
All sourcesNASA NTRSDOE OSTI
NASA NTRS1990

Characterization and distribution of a maize cDNA encoding a peptide similar to the catalytic region of second messenger dependent protein kinases

Maize (Zea mays) roots respond to a variety of environmental stimuli which are perceived by a specialized group of cells, the root cap. We are studying the transduction of extracellular signals by roots, particularly the role of protein kinases. Protein phosphorylation by kinases is an important step in many eukaryotic signal transduction pathways. As a first phase of this research we have isolated a cDNA encoding a maize protein similar to fungal and animal protein kinases known to be involved in the transduction of extracellular signals. The deduced sequence of this cDNA encodes a polypeptide containing amino acids corresponding to 33 out of 34 invariant or nearly invariant sequence features characteristic of protein kinase catalytic domains. The maize cDNA gene product is more closely related to the branch of serine/threonine protein kinase catalytic domains composed of the cyclic-nucleotide- and calcium-phospholipid-dependent subfamilies than to other protein kinases. Sequence identity is 35% or more between the deduced maize polypeptide and all members of this branch. The high structural similarity strongly suggests that catalytic activity of the encoded maize protein kinase may be regulated by second messengers, like that of all members of this branch whose regulation has been characterized. Northern hybridization with the maize cDNA clone shows a single 2400 base transcript at roughly similar levels in maize coleoptiles, root meristems, and the zone of root elongation, but the transcript is less abundant in mature leaves. In situ hybridization confirms the presence of the transcript in all regions of primary maize root tissue.

NASA Program Space Biology↗
DOE OSTI2021

SCOPe: improvements to the structural classification of proteins – extended database to facilitate variant interpretation and machine learning

Abstract The Structural Classification of Proteins—extended (SCOPe, https://scop.berkeley.edu) knowledgebase aims to provide an accurate, detailed, and comprehensive description of the structural and evolutionary relationships amongst the majority of proteins of known structure, along with resources for analyzing the protein structures and their sequences. Structures from the PDB are divided into domains and classified using a combination of manual curation and highly precise automated methods. In the current release of SCOPe, 2.08, we have developed search and display tools for analysis of genetic variants we mapped to structures classified in SCOPe. In order to improve the utility of SCOPe to automated methods such as deep learning classifiers that rely on multiple alignment of sequences of homologous proteins, we have introduced new machine-parseable annotations that indicate aberrant structures as well as domains that are distinguished by a smaller repeat unit. We also classified structures from 74 of the largest Pfam families not previously classified in SCOPe, and we improved our algorithm to remove N- and C-terminal cloning, expression and purification sequences from SCOPe domains. SCOPe 2.08-stable classifies 106 976 PDB entries (about 60% of PDB entries).

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Overexpression of a Sugarcane BAHD Acyltransferase Alters Hydroxycinnamate Content in Maize Cell Wall

The purification of hydroxycinnamic acids [ p -coumaric acid ( p CA) and ferulic acid (FA)] from grass cell walls requires high-cost processes. Feedstocks with increased levels of one hydroxycinnamate in preference to the other are therefore highly desirable. We identified and conducted expression analysis for nine BAHD acyltransferase ScAts genes from sugarcane. The high conservation of AT10 proteins, together with their similar gene expression patterns, supported a similar role in distinct grasses. Overexpression of ScAT10 in maize resulted in up to 75% increase in total p CA content. Mild hydrolysis and derivatization followed by reductive cleavage (DFRC) analysis showed that p CA increase was restricted to the hemicellulosic portion of the cell wall. Furthermore, total FA content was reduced up to 88%, resulting in a 10-fold increase in the p CA/FA ratio. Thus, we functionally characterized a sugarcane gene involved in p CA content on hemicelluloses and generated a C4 plant that is promising for valorizing p CA production in biorefineries.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Evaluation of near-ambient algal biomass fractionation conditions for bioproduct development

This contribution describes an algal fractionation scheme based on cell lysing and carbohydrate hydrolysis under acidic conditions, coupled with solvent extraction, that produces algal lipids, carbohydrates, and proteinaceous solid from partially dewatered algal biomass. Here, a design of experiments analysis was employed to identify the effect of fractionation conditions on the yields of the three product streams. By selection of appropriate conditions, the process can be steered from simple lipid extraction to near complete fractionation of the biomass. Lipid purification and upgrading were respectively achieved with a low-cost adsorbent and an inexpensive Ni-based catalyst that deoxygenated the lipids via decarboxylation/decarbonylation, an approach offering several advantages over the hydrodeoxygenation-based processes typically employed to convert lipids to hydrocarbons. The proteinaceous solids obtained were found to have much lower ash content as well as higher protein content relative to the untreated algae, enhancing the suitability of this material as a feedstock for the production of bioplastics.

09 BIOMASS FUELS↗
NASA NTRS1980

A New Image for the Water Hyacinth

Walt Disney Company activated a sewage treatment plan using NASA technology to create water hyacinths to clean wastewater by absorbing and metabolizing pollutants. Plants have exciting promise as a natural water purification system which can be established at a fraction of the cost of a conventional sewage treatment facility. Harvested plants can be used as fertilizer. They can also be heat-treated to produce consumer energy in the form of methane gas. If an economical method of drying plants can be developed they may find further utility as high protein animal feed.

Source record↗
NASA NTRS1998

Co- and/or post-translational modifications are critical for TCH4 XET activity

TCH4 encodes a xyloglucan endotransglycosylase (XET) of Arabidopsis thaliana. XETs endolytically cleave and religate xyloglucan polymers; xyloglucan is one of the primary structural components of the plant cell wall. Therefore, XET function may affect cell shape and plant morphogenesis. To gain insight into the biochemical function of TCH4, we defined structural requirements for optimal XET activity. Recombinant baculoviruses were designed to produce distinct forms of TCH4. TCH4 protein engineered to be synthesized in the cytosol and thus lack normal co- and post-translational modifications is virtually inactive. TCH4 proteins, with and without a polyhistidine tag, that harbor an intact N-terminus are directed to the secretory pathway. Thus, as predicted, the N-terminal region of TCH4 functions as a signal peptide. TCH4 is shown to have at least one disulfide bond as monitored by a mobility shift in SDS-PAGE in the presence of dithiothreitol (DTT). This disulfide bond(s) is essential for full XET activity. TCH4 is glycosylated in vivo; glycosidases that remove N-linked glycosylation eliminated 98% of the XET activity. Thus, co- and/or post-translational modifications are critical for optimal TCH4 XET activity. Furthermore, using site-specific mutagenesis, we demonstrated that the first glutamate residue of the conserved DEIDFEFL motif (E97) is essential for activity. A change to glutamine at this position resulted in an inactive protein; a change to aspartic acid caused protein mislocalization. These data support the hypothesis that, in analogy to Bacillus beta-glucanases, this region may be the active site of XET enzymes.

NASA Discipline Cell Biology↗
DOE OSTI2021

2-Aminoimidazole reduces fouling and improves membrane performance

Biofouling is difficult to control and hinders the performance of membranes in all applications but is of particular concern when natural waters are purified. Fouling, via multiple mechanisms (organic-only, biofouling-only, cell-deposition-only, and organic + biofouling), of a commercially available membrane (control) and a corresponding membrane coated with an anti-biofouling 2-aminoimidazole (2-AI membrane) was monitored and characterized during the purification of a natural water. Results show that the amount of bacterial cell deposition and organic fouling was not significantly different between control and 2-AI membranes; however, biofilm formation, concurrent or not with other fouling mechanisms, was significantly inhibited (95–98%, p < 0.001) by the 2-AI membrane. The limited biofilm that formed on the 2-AI membrane was weaker (as indicated by the polysaccharide to protein ratio) and thus presumably easier to remove. The conductivity rejection by the 2-AI and control membranes was not significantly different throughout the 75-h experiments, but the rejection of dissolved organic carbon by biofouled (biofouling-only, cell-deposition-only, and organic + biofouling) 2-AI membranes was statistically higher (10–12%, p = 0.003–0.07). When biofouled, the water permeance of the 2-AI membranes decreased significantly less (p < 0.05) over 75 hours than that of the control membranes, whether or not other additional types of fouling occurred concurrently. Despite the initially lower water permeances of 2-AI membranes (11% lower on average than controls), the 2-AI membranes outperformed the controls (10–11% higher average water permeance) after biofilm formation occurred. Overall, 2-AI membranes fouled less than controls without detriment to water productivity and solute rejection.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Biomanufacturing and bioprocessing of lunar regolith

Microbial biomanufacturing is important to accelerate lunar construction because it can leverage lunar material and waste streams as feedstocks to create a circular production system. In-space bio-mining and biomanufacturing using moon and asteroidal source material will enable the creation of infrastructure, produce industrial fuels and lubricants, and enable recovery of actinides and rare-earth elements (REEs) present in trace concentrations. Moreover, biomanufacturing in closed-loop systems (recycling and reuse of resources toward the establishment of a circular economy) will enable long-term lunar activities by recycling waste (CO 2 , gray water) and producing oxygen and biomaterials. Our response focuses on the use of lunar regolith and waste streams as feedstocks for protein and microbial-enabled biomining and bioprocessing to extract actinides and REEs, and to create biocomposites for lunar infrastructure. We envision an enclosed process that initiates with (1a) extracting actinides and REEs from lunar regolith using immobilized proteins, followed by (1b) creating biocomposites from the post-extracted lunar regolith for infrastructure, and (1c) cultivating diatoms and other microalgae on waste streams to harvest silica shells for incorporating into biocomposites and to generate O 2 for human respiration and/or producing refinable feedstocks. LLNL has significant expertise in all three processes and provides facilities, personnel, and expertise at the intersection of metal (lanthanide, actinide, transition) separations, purifications, biohydrometallurgy, radiobiochemistry, synthetic and systems biology, and materials science and engineering. Importantly, all three processes are relatively well-studied for Earth-based workflows and can be derisked for demonstration on the lunar surface by 2029.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS1984

Bioassay, isolation and studies on the mechanism of action of neurite extension factor

The identification and purification of molecules active in promoting neurite outgrowth requires a sensitive reproducible bioassay. A quantitative bioassay was utilized to purify a neurite extension factor (NEF) based on counting the number of phase bright neurons with processes at least equal to one cell body diameter after 20 hrs. in culture is defined, serum free medium. Using a combination of heat treatment DEAE cellulose chromatography and gel filtration, an acidic protein of M sub r = 75,000 was highly purified. Upon reduction, it yields subunits of M sub r = 37,000. Purified fractions are active half maximally at 100 ng/ml in inducing neurite outgrowth in this bioassay. Currently, monoclonal antibodies to NEF are being produced. Female Balb C mice were immunized with the antigen and fusions with mouse myeloma cells will be performed to yield hybridoma cells.

Kligman, D.↗
NASA NTRS2011

Assessment of Resistance of Bacillus Horneckiae Endospores to UV Radiation and Function of Their Extraneous Layer in Resistance

Spore-forming microbes are highly resistant to various physical and chemical conditions, which include ionizing and UV radiation, desiccation and oxidative stress, and the harsh environment of outer space or planetary surfaces. The spore's resistance might be due to their metabolically dormant state, and/or by the presence of a series of protective structures that encase the interior-most compartment, the core, which houses the spore chromosome. These spores have multiple layers surrounding the cell that are not found in vegetative cells, and some species have an outer layer of proteins and glycoproteins termed the "exosporium" or a fibrous "extraneous layer" (EL). Bacillus horneckiae is an EL-producing novel sporeformer isolated from a Phoenix spacecraft assembly clean room, and it has previously demonstrated resistance to UV radiation up to 1000 J/m(sup 2). The EL appears to bind B. horneckiae spores into large aggregations, or biofilms, and may confer some UV resistance to the spores. Multiple culturing and purification schemes were tried to achieve high purity spores because vegetative cells would skew UV resistance results. An ethanol-based purification scheme produced high purity spores. Selective removal of the EL from spores was attempted with two schemes: a chemical extraction method and physical extraction (sonication). Results from survival rates in the presence and absence of the external layer will provide a new understanding of the role of biofilms and passive resistance that may favor survival of biological systems in aggressive extra-terrestrial environments. The chemical extraction method decreased viable counts of spores and lead to an inconclusive change UV resistance relative to non-extracted spores. The physical extraction method lead to non-aggregated spores and did not alter viability; however, it produced UV resistance profiles similar to non-extracted spores. In addition to the EL-removal study, samples of B. horneckiae spores dried on aluminum coupons and exposed to increasing UV (200-400 nm range) levels (0 to 8.0 x 105 kJ/m(sup 2)) were tested for viability, which indicated that the maximum UV exposure level that still resulted in viable spores was 5.0 x 10? kJ/m(sup 2).

resistance↗
NASA NTRS2006

Fluorescent Approaches to High Throughput Crystallography

We have shown that by covalently modifying a subpopulation, less than or equal to 1%, of a macromolecule with a fluorescent probe, the labeled material will add to a growing crystal as a microheterogeneous growth unit. Labeling procedures can be readily incorporated into the final stages of purification, and the presence of the probe at low concentrations does not affect the X-ray data quality or the crystallization behavior. The presence of the trace fluorescent label gives a number of advantages when used with high throughput crystallizations. The covalently attached probe will concentrate in the crystal relative to the solution, and under fluorescent illumination crystals show up as bright objects against a dark background. Non-protein structures, such as salt crystals, will not incorporate the probe and will not show up under fluorescent illumination. Brightly fluorescent crystals are readily found against less bright precipitated phases, which under white light illumination may obscure the crystals. Automated image analysis to find crystals should be greatly facilitated, without having to first define crystallization drop boundaries as the protein or protein structures is all that shows up. Fluorescence intensity is a faster search parameter, whether visually or by automated methods, than looking for crystalline features. We are now testing the use of high fluorescence intensity regions, in the absence of clear crystalline features or "hits", as a means for determining potential lead conditions. A working hypothesis is that kinetics leading to non-structured phases may overwhelm and trap more slowly formed ordered assemblies, which subsequently show up as regions of brighter fluorescence intensity. Preliminary experiments with test proteins have resulted in the extraction of a number of crystallization conditions from screening outcomes based solely on the presence of bright fluorescent regions. Subsequent experiments will test this approach using a wider range of proteins. The trace fluorescently labeled crystals will also emit with sufficient intensity to aid in the automation of crystal alignment using relatively low cost optics, further increasing throughput at synchrotrons.

Pusey, Marc L.↗
NASA NTRS1997

Composition of the carbohydrate granules of the cyanobacterium, Cyanothece sp. strain ATCC 51142

Cyanothece sp. strain ATCC 51142 is an aerobic, unicellular, diazotrophic cyanobacterium that temporally separates O2-sensitive N2 fixation from oxygenic photosynthesis. The energy and reducing power needed for N2 fixation appears to be generated by an active respiratory apparatus that utilizes the contents of large interthylakoidal carbohydrate granules. We report here on the carbohydrate and protein composition of the granules of Cyanothece sp. strain ATCC 51142. The carbohydrate component is a glucose homopolymer with branches every nine residues and is chemically identical to glycogen. Granule-associated protein fractions showed temporal changes in the number of proteins and their abundance during the metabolic oscillations observed under diazotrophic conditions. There also were temporal changes in the protein pattern of the granule-depleted supernatant fractions from diazotrophic cultures. None of the granule-associated proteins crossreacted with antisera directed against several glycogen-metabolizing enzymes or nitrogenase, although these proteins were tentatively identified in supernatant fractions. It is suggested that the granule-associated proteins are structural proteins required to maintain a complex granule architecture.

NASA Discipline Cell Biology↗
NASA NTRS1974

Electrophoresis in space at zero gravity

Early planning for manufacturing operations in space include the use of electrophoresis for purification and separation of biological materials. Greatly simplified electrophoresis apparatus have been flown in the Apollo 14 and 16 missions to test the possibility of stable liquid systems in orbit. Additionally, isoelectric focusing and isotachophoresis are of particular interest as they offer very high resolution and have self-sharpening boundaries. The value of possible space electrophoresis is substantial. For example, present technology permits large fractionation of only a few of blood proteins many fractions, and separated cell populations are needed for research.

Bier, M.↗
DOE OSTI2023

Analysis of a macrophage carbamylated proteome reveals a function in post-translational modification crosstalk

Background. Lysine carbamylation is a biomarker of rheumatoid arthritis and kidney diseases. However, its cellular function is understudied due to the lack of tools for systematic analysis of this post-translational modification (PTM). Methods. We adapted a method to analyze carbamylated peptides by co-affinity purification with acetylated peptides based on the cross-reactivity of anti-acetyllysine antibodies. We also performed immobilized-metal affinity chromatography to enrich for phosphopeptides, which allowed us to obtain multi-PTM information from the same samples. Results. By testing the pipeline with RAW 264.7 macrophages treated with bacterial lipopolysaccharide, 7,299, 8,923 and 47,637 acetylated, carbamylated, and phosphorylated peptides were identified, respectively. Our analysis showed that carbamylation occurs on proteins from a variety of functions on sites with similar as well as distinct motifs compared to acetylation. To investigate possible PTM crosstalk, we integrated the carbamylation data with acetylation and phosphorylation data, leading to the identification 1,183 proteins that were modified by all 3 PTMs. Among these proteins, 54 had all 3 PTMs regulated by lipopolysaccharide and were enriched in immune signaling pathways, and in particular, the ubiquitin-proteasome pathway. We found that carbamylation of linear diubiquitin blocks the activity of the anti-inflammatory deubiquitinase OTULIN. Conclusions. Overall, our data show that anti-acetyllysine antibodies can be used for effective enrichment of carbamylated peptides. Moreover, carbamylation may play a role in PTM crosstalk with acetylation and phosphorylation, and that it is involved in regulating ubiquitination in vitro.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS1999

Regiochemical control of monolignol radical coupling: a new paradigm for lignin and lignan biosynthesis

BACKGROUND: Although the lignins and lignans, both monolignol-derived coupling products, account for nearly 30% of the organic carbon circulating in the biosphere, the biosynthetic mechanism of their formation has been poorly understood. The prevailing view has been that lignins and lignans are produced by random free-radical polymerization and coupling, respectively. This view is challenged, mechanistically, by the recent discovery of dirigent proteins that precisely determine both the regiochemical and stereoselective outcome of monolignol radical coupling. RESULTS: To understand further the regulation and control of monolignol coupling, leading to both lignan and lignin formation, we sought to clone the first genes encoding dirigent proteins from several species. The encoding genes, described here, have no sequence homology with any other protein of known function. When expressed in a heterologous system, the recombinant protein was able to confer strict regiochemical and stereochemical control on monolignol free-radical coupling. The expression in plants of dirigent proteins and proposed dirigent protein arrays in developing xylem and in other lignified tissues indicates roles for these proteins in both lignan formation and lignification. CONCLUSIONS: The first understanding of regiochemical and stereochemical control of monolignol coupling in lignan biosynthesis has been established via the participation of a new class of dirigent proteins. Immunological studies have also implicated the involvement of potential corresponding arrays of dirigent protein sites in controlling lignin biopolymer assembly.

NASA Discipline Plant Biology↗
NASA NTRS1988

Rapid changes in protein phosphorylation associated with light-induced gravity perception in corn roots

The effect of light and calcium depletion on in vivo protein phosphorylation was tested using dark-grown roots of Merit corn. Light caused rapid and specific promotion of phosphorylation of three polypeptides. Pretreatment of roots with ethylene glycol bis N,N,N',N' tetraacetic acid and A23187 prevented light-induced changes in protein phosphorylation. We postulate that these changes in protein phosphorylation are involved in the light-induced gravity response.

NASA Program Space Biology↗
NASA NTRS1992

Two endogenous proteins that induce cell wall extension in plants

Plant cell enlargement is regulated by wall relaxation and yielding, which is thought to be catalyzed by elusive "wall-loosening" enzymes. By employing a reconstitution approach, we found that a crude protein extract from the cell walls of growing cucumber seedlings possessed the ability to induce the extension of isolated cell walls. This activity was restricted to the growing region of the stem and could induce the extension of isolated cell walls from various dicot stems and the leaves of amaryllidaceous monocots, but was less effective on grass coleoptile walls. Endogenous and reconstituted wall extension activities showed similar sensitivities to pH, metal ions, thiol reducing agents, proteases, and boiling in methanol or water. Sequential HPLC fractionation of the active wall extract revealed two proteins with molecular masses of 29 and 30 kD associated with the activity. Each protein, by itself, could induce wall extension without detectable hydrolytic breakdown of the wall. These proteins appear to mediate "acid growth" responses of isolated walls and may catalyze plant cell wall extension by a novel biochemical mechanism.

Non-NASA Center↗